Project Summary/Abstract The human airway suffers genetically determined dysfunctions, such as cystic fibrosis, or environmentally acquired diseases, like chronic obstructive pulmonary disease. The airway epithelium is characterized by mucus and fluid secretion, regulation of ion homeostasis, and the presence of multiciliated cells, which generate a directional movement of fluid to transport inhaled particles and pathogens out of the respiratory tract. The apical surface of multiciliated cells is decorated with dozens of motile cilia, that beat synchronously to generate this extracellular fluid flow. Defects arising from impaired motile cilia function or abnormal secretion making these patients susceptible to airway infections. Proton pump inhibitors belong to the most widely prescribed drugs and are often given to hospitalized patients, but increased rates of pneumonia were reported. As multi-resistant pathogens are more frequently found in hospital settings, patients treated with proton pump inhibitors are at high risk. In order to improve the current state of diagnosis, treatment and preventive care of airway diseases, including infections, it is critical t understand how this important barrier against pathogens is established and maintained at the tissue, cell and molecular level. This project will establish an experimental pipeline to efficienty test the effects of signaling pathways, mutations, drugs and pathogens on vertebrate mucociliary epithelia in a comparative manner. In addition to established models (primary human cell culture and mouse airways), I will take advantage of two emerging systems, i.e. the Xenopus embryonic epidermis and immortalized human basal cells (BCi-NS1.1). The Xenopus embryonic epidermis has proven to be an excellent model to study the biology of vertebrate mucociliary epithelia, as it develops fast, is easily accessible to manipulation and analysis, and molecular functions of signaling pathways and transcriptional regulators are conserved between Xenopus and mammals. BCi-NS1.1 cells can be differentiated into mucociliary cells in culture, like primary human airway epithelial cells, but are genetically manipulable. Using these additional emerging models will facilitate new applications to investigate molecular mechanisms in airway mucociliary epithelia. First, this project will resolve the homology of mucociliary cell types across the experimental systems, and establish stable genetically manipulated lines using human BCi-NS1.1 cells. Next, roles of Wnt signaling in mucociliary cell specification, morphogenesis and function will be investigated. Although Wnt signaling is required for normal mucociliary epithelial development and function in Xenopus and mammals, it remains unresolved which cell types require Wnt signaling during which steps of specification and differentiation. The Wnt signaling-dependent transcriptional network will be analyzed to determine functions in specification, ciliogenesis and secretion. Wnt signaling activation is als pH-dependent and we have found that loss of proton transporters affects gene expression and cell differentiation in mucociliary epithelia. Together with data on the roles of Wnt signaling, experiments manipulating proton pump function will allow me to experimentally address the underlying molecular mechanisms of proton pump dysfunction-associated pneumonia in human patients. In summary, I will use the mucociliary epidermis of Xenopus embryos, in combination with mouse models and cultured human airway cells to resolve homology of mucociliary cell types, functions of the Wnt transcriptional network in mucociliary epithelia, and how transmembrane proton pumps contribute to signaling regulation and respiratory health. The results from this project will expand applications in emerging model systems for airway research, broaden our knowledge of molecular cell signaling mechanisms in mucociliary development and function, and resolve how proton pumps contribute to mucociliary clearance.